investigations in tuberculosis and advances
TRANSCRIPT
![Page 1: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/1.jpg)
Investigations in Mycobacterium Tuberculosis and Advances
Dr. Nirish VaidyaResident, First yearInternal Medicine
KUSMS, Kathmandu University Hospital
![Page 2: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/2.jpg)
2
Mycobacterium tuberculosis-Characteristics
•Gram positive •Obligate aerobe•Non-spore-forming•Non-motile rod•Mesophile•Slow generation time: 15-20 hours
•May contribute to virulence
•Lipid rich cell wall contains mycolic•Responsible for many of this bacterium’s characteristic properties
![Page 3: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/3.jpg)
The Burden• Worldwide, 9.6 million people are estimated to have fallen ill with TB in
2014• In 2014, 6 million new cases of TB were reported to WHO, fewer than
two-thirds (63%) of the 9.6 million people estimated to have fallen sick with the disease. This means that worldwide, 37% of new cases went undiagnosed or were not reported.
• Of the 480 000 cases of multidrug-resistant TB (MDR-TB) estimated to have occurred in 2014, only about a quarter of these – 123 000 – were detected and reported
• Delay in the diagnosis?• Delay in getting result?• To reduce this burden, detection and treatment gaps must be
addressed, funding gaps closed and new tools developed• Laboratory methods play a crucial
![Page 4: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/4.jpg)
TECHNIQUES
1. Detection of Mycobacterium2. Molecular Methods3. Identifications of Species4. Immunological Methods
![Page 5: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/5.jpg)
Sputum Microscopy• Carbol fuchsin • Fluorochrome stain such as auramine-rhodamine– Improves the sensitivity of Mtb detection– Recent advances in light-emitting diode (LED)
technology have widened the applicability of fluorescent microscopy
• Persons unable to cough up sputum– induce sputum– bronchoscopy – gastric aspiration The quantity of sputum (at least 5 mL)
![Page 6: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/6.jpg)
Sputum smears stained by Z-N stain
Advantage: - cheap – rapid - Easy to perform - High predictive value > 90%
-Specificity of 98%Disadvantages: - sputum ( need to contain 5000-10000 AFB/ ml.)
Sensitivity: 40-70% - Young children, elderly & HIV infected persons
may not produce cavities & sputum containing AFB.Solution:
-Centrifused sample-Overnight Sedimentation
![Page 7: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/7.jpg)
7
New Policy and Smear microscopy definition of a TB case
• New definition in 2007 (ISTC):“person with al one AFB is sufficient out of a total of two examined”
• 2 samples regardless the collection time– Retrospective study
• Nelson, SM, Deike, MA, Cartwright, CP. Value of examining multiple sputum specimens in the diagnosis of pulmonary tuberculosis. J Clin Microbiol 1998; 36:467. – overall, 92 percent of cases would have been detected with two specimens
• Craft, DW, Jones, MC, Blanchet, CN, et al. Value of examining three acid-fact bacillus sputum smears for the removal of patients suspected of having tuberculosis from the "airborn precautions" category. J Clin Microbiol 2000; 38:4285. – a third sputum smear was of no additional value
*
7
![Page 8: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/8.jpg)
Interpretation of sputum stained by Z N Stain (WHO )
No No AFB/300 OIF Negative
1-9 No of AFB/100 OIF Mention the number
10-99 No of AFB/100 OIF 1+
1-10 No ofAFB/50 OIF 2+
>10 No of AFB/ 20 OIF 3+
![Page 9: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/9.jpg)
The smear may be stained by auramine-O dye. TB bacilli are stained yellow against dark
background & easily visualized using florescent microscope.
Advantages: - More sensitive - RapidDisadvantages: - Hazards of dye toxicity - More expensive
LED Fluorescence Microscopy
![Page 10: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/10.jpg)
Culture
• GOLD STANDARD• Solid media: Lowenstein-Jensen– Brown, granular colonies "buff, rough and tough").– Four weeks, due to the slow doubling
• Liquid media: used in commercially available automated systems
• Used to confirm diagnosis of TB
![Page 11: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/11.jpg)
Cultures• Sensitivity: 80-85%• Specificity: 98%• Times needed:
– Solid medium• 4-8 wks
– Liquid medium• 2 wks
Advantages: - More sensitive (need lower no. of bacilli 10-100 / ml) -Differentiate between TB complex & NTM using biochemical
reactions - Sensitivity tests for antituberculous drugs Disadvantages: Slowly growing ( up to 8 weeks ) in solid medium
![Page 12: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/12.jpg)
AFB smear vs. Cultures
• AFB smear– Need 5000-10000/ml bacilli– Rapid diagnosis
• Cultures– Need lower no.10-100/ml bacilli– More sensitive – Allows drug sensitivity test
![Page 13: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/13.jpg)
Screening-Tuberculin test ( Mantoux test)
• Delayed HSR against tubercular protein
• Inject intradermally 0.1 ml of 5TU PPD tuberculin
• Read reaction 48-72 hours after injection
• Measure only induration• Record reaction in mm
13
![Page 14: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/14.jpg)
Classifying the tuberculin reaction
• >5 mm is classified as positive in– HIV-positive persons– Recent contacts of TB case– Persons with fibrotic changes on CXR consistent
with old healed TB– Patients with organ transplants and other
immunosuppressed patients
![Page 15: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/15.jpg)
Classifying the tuberculin reaction
• >10 mm is classified as positive in– Recent arrivals from high-prevalence countries– Injection drug users– Residents and employees of high-risk settings– Mycobacteriology laboratory personnel– Persons with clinical conditions that place them at
high risk– Children <4 years, or children and adolescents
exposed to adults in high-risk categories
![Page 16: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/16.jpg)
Classifying the tuberculin reaction
• >15 mm is classified as positive in– Persons with no known risk factors for TB
![Page 17: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/17.jpg)
Tuberculin Test: Interpretation
• A positive test
• The person’s body was infected with TB bacteria• Latent TB infection or TB disease??-Additional test needed• may develop reactivation type of T.B.
• A negative tuberculin test
• excludes infection in suspected persons • The person’s body did not react to the test, and that latent
TB infection or TB disease is not likely.• are at risk of gaining new infection
17
![Page 18: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/18.jpg)
False negative
•Severe tuberculosis infection (Miliary T.B.) - Hodgkin’s disease • Corticosteroid therapy -
Malnutrition - AIDS• Children below 5 years of
age with no exposure history
False positive
• Atypical mycobacterium• BCG vaccination
– Usually <10 mm by adulthood
18
Recombinant Early Secretory antigenic target (ESAT)-6 instead of tuberculin have demonstrated safety and tolerability of such a test. May solve the problem but is on Phase I trial
![Page 19: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/19.jpg)
Newer techniques
![Page 20: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/20.jpg)
Septi-check AFB• Simultaneous detection of
Mycobacterium tuberculosis and non tuberculosis mycobacterial
• Requires ~ 3 weeks of incubation• Specimens: Sputum,
bronchoalveolar lavage, urine, stool, body fluids, biopsy tissues, wounds and skin
• Advantage over convetions Mb isolation media-Small inocula-Short time
![Page 21: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/21.jpg)
Microcolony detection on solid media(MODS)
• Uses simple light microscopy on plates with a thin layer of 7H11 agar medium
• Identifies characteristics strings and tangles of M.tb
• Rapid yield ~9-10 days• Low cost alternate for
detecting drug resistance• Sensitivity 92%• High technical skills,
biosafety cabins complex agar medium
![Page 22: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/22.jpg)
Mycobacteria Growth Indicator Tube (MGIT)
Tube contains modified Middlebrook 7H9 broth base
All types of clinical specimens, pulmonary as well as extra-pulmonary ( except blood) could be cultured
![Page 23: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/23.jpg)
BACTEC • Specimen is inoculated in 14C labeled
7H12 medium(MGIT)• Incubated• Mycobacterium multiply generating
14Co2, increase fluorescence in sensor of the bottle
• The instrument scans the MGIT every 60 minutes for increased fluorescence.
• An instrument-positive tube contains approximately 10^5 to 10^6 CFU/mL.
• Can detect growth as early as 4 days and drug susceptibility in 21-28 days.
• Cost effective
![Page 24: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/24.jpg)
MB/Bac T• Non-radiometric continuous monitoring
system that utilizes colorimetric sensor and reflected light to continuously monitor the Co2 concentration in culture medium
• Good alternative to BACTEC– But takes slightly longer time ~15-20 days
• The mean time to detection was 12.9 days by BACTEC MGIT960, and 15.0 days with BACTEC 460,compared with 27.0 days LJ Medium (Advances in the diagnosis of tuberculosis; C LANGE et al;Repirology)
![Page 25: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/25.jpg)
ESP Blood culture system
• Fully automated continuous monitoring culture system by evaluating gas consumption by microbes rather than production of CO2 as in BACTEC
• Mean time of detection 11-16 days
![Page 26: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/26.jpg)
26
• Detection and Diagnosis• – uncultivable or difficult to culture
• – need for rapid answer• Prognosis and management• – need for quantitative
information (viral load)• – susceptibility testing (drug
resistance) without culture• • Molecular resistance
testing
Molecular DiagnosticsWhy?
![Page 27: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/27.jpg)
Nucleic Acid Amplification (NAA)
• Both detection and identification of M. tuberculosis through enzymatic amplification of bacterial DNA
• Amplify M. tuberculosis-specific nucleic acid sequences using a nucleic acid probe.
• Used mostly for confirmation of smear positive results
• Quick results, diagnostic accuracy• Require as few as 1O bacilli from a given sample• Expensive
![Page 28: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/28.jpg)
Nucleic Acid Amplification (NAA)
• Specificity in the range of 98% to 99%. • Useful technology for rapid diagnosis of smear
negative cases of active TB• Able to identify 50-60% of smear -ve culture +ve cases• Distinguish M.tb from NTM in smear +ve cases• Should not be used to replace sputum microscopy as
an initial screen & culture remains the gold standard• Very high degree of quality control required
![Page 29: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/29.jpg)
Polymerase Chain Reaction (PCR)
• Allows sequences of DNA to be amplified in vitro
• Detection is based on multiplication not of whole bacilli, as in culture, but of their genetic material, chromosomal DNA or ribosomal RNA, typical for MTB or MTB complex.
• Most common target used for PCR is insertion sequence IS6110 which is specific for M. tb.
![Page 30: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/30.jpg)
30
Advantages: - Rapid procedure ( 3 – 4
hours) - High sensitivity (1-10 bacilli / ml sputum)Sensitivity ~95%, Specificity
~100%Disadvantage
-High cost
Polymerase Chain Reaction (PCR)
![Page 31: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/31.jpg)
31
Xpert MTB/RIF • Real time PCR assay to amplify a specific
sequence of the rpo B gene• Xpert MTB/RIF detects M. tuberculosis as well
as rifampicin resistance• Cartridge based Automated molecular test
M.Tb and its resistance to Rifampin
![Page 32: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/32.jpg)
![Page 33: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/33.jpg)
33
Advantages with GeneXpert
• Simultaneous detection of both MTB and rifampicin resistance, a marker for MDR strains
• Unprecedented sensitivity for detecting MTB — even in smear negative, culture positive specimens
• Results in two hours; requires no instrumentation other than the GeneXpert® System
• On-demand results enable physicians to treat rapidly and effectively
•
![Page 34: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/34.jpg)
• Monoresistance to Rifampicin ~5 %• Concurrent resistance with Isoniazide ~95%– Dx of MDR Tb with high level of accuracy
• Xpert MTB/RIF has higher sensitivity for Sputum smear positive cases than Smear negative cases. Nontheless a valuable tool for smear negative cases
• International standard for TB Care recommended Xpert MTB/RIF assay with Culture for Sputum Negative Suspected cases
![Page 35: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/35.jpg)
Chromatography
• Identification of different species• Used in reference labs for epidemiologic
studies• Results within 2 hours• High cost
![Page 36: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/36.jpg)
TB PNA FISH
• Fluorescence in situ hybridization (FISH) using specific Peptide nucleic acid (PNA) probe allows differentiation between tuberculous and nontuberculous mycobacteria in AFB+ve cultures
• Specificity and predictive values approaches 100%
![Page 37: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/37.jpg)
37
• DNA amplification technique• It is a variant of PCR, in which a
pair of oligonucleotides are made to bind to one of the DNA target strands, so that they are adjacent to each other. A second pair of oligonucleotides is designed to hybridize to the same regions on the complementary DNA.
Ligase Chain Reaction
![Page 38: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/38.jpg)
38
• The action of DNA polymerase and ligase in the presence of nucleotides results in the gap between adjacent primers being filled with appropriate nucleotides and ligation of primers
• Mainly being used for respiratory sample
• High overall specificity and sensitivity for smear +ve and –ve specimens.
Ligase Chain Reaction
![Page 39: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/39.jpg)
39
Loop-mediated isothermal amplification (LAMP)
• The mixing of all reagent in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63°C 60-min incubation time.
• It is a novel nucleic acid amplification method• Used for detection of M.tb complex, M.avium, and
M.intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT) or on a solid medium (Ogawa’s medium).
![Page 40: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/40.jpg)
Interferon-gamma release assays (IGRA)
• Measure interferon (IFN-γ) released by sensitized T cells after stimulation by M. tuberculosis antigens. Measures immune reactivity to M.tb.
• The test uses synthetic peptide antigens (ESAT-6,CFP-10) that simulate this protein to generate the immune response
• No memory as in Mantoux, but tells us it’s a RECENT INFECTION
![Page 41: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/41.jpg)
Available Interferon-gamma release assays (IGRA) tests
Quantiferon Gold T-SPOT.TB
Limitation is COST. NRs 4000/-
![Page 42: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/42.jpg)
Advantages of IGRA
• Quantitative reports– postive or negative
• Result in single patient visit• Not affected by BCG status and NTB• Not affected by repeated IGRA
![Page 43: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/43.jpg)
Disadvantages of IGRA
• Does not tell Latent or active TB• COST• Results altered in immune compromised
![Page 44: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/44.jpg)
Monocyte-amplified INF-γ release assays (MIGRAS)
• Principle:– INF release leads to subsequent release of INF-
responsive chemokines such as MIG and IP-10– Measures this chemokines
![Page 45: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/45.jpg)
Transcription mediated amplification (TMA)
• Can identify the presence of genetic information unique to M. tuberculosis directly from pre processed clinical specimens
• Sensitivity ~ culture• Sensitivity ~96% and Specificity 100%• Disadvantage– Positive results in both viable and dead bacilli
![Page 46: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/46.jpg)
Other approaches
• Detection of anti-mycobacterium superoxide dismutase antibodies
• MPB 64 patch test– Transdermal patch containing MPB 64, an antigen
specific for M tuberculosis – Results obtained d after 3-4 days– Sensitivity 97.8% and Specificity 100%– Discriminated latent infection from active disease
![Page 47: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/47.jpg)
FAST Plaque TB
• Specific mycobacteriophages infect M.tb in specimen
• Intracellular phages undergo replications with subsequent release of phages from host cells
• Released phages are incubated along with other nonpathogenic organisms in agar plate – produces zone of clearing (plaques) – POSITIVE
• Rapid <24 hours
![Page 48: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/48.jpg)
FAST-Plaque-Response
• Extention of FAST-Plaque TB• Allows early detection of Rifampicin resistance
![Page 49: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/49.jpg)
Adenosine Deaminase (ADA)
• Surrogate marker in pleural, pericardial and peritoneal fluids and CSF
• Sensitivity of 100%, Specificity 94.6% (study performed in India)
![Page 50: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/50.jpg)
Detection of lipoarabinomanan
• LAM is a cell lipopolysaccharide Ag of M tb• LAM-ELISA assay in urinary useful in HIV
associated tuberculosis with low CD4 counts• Sensitivity 93%, Specificity 95%• Useless in Blood TB-ELISA
![Page 51: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/51.jpg)
Dr.T.V.Rao MD 51
![Page 52: Investigations in Tuberculosis and advances](https://reader033.vdocuments.mx/reader033/viewer/2022052418/58ede8211a28ab54088b4577/html5/thumbnails/52.jpg)
Thank you